G C A T

T A C G
G C A T
genes
Review
The Aging Mitochondria
Pierre Theurey 1, * and Paola Pizzo 1,2, * ID

1 Department of Biomedical Sciences, University of Padova, Padova 35121, Italy

2 Neuroscience Institute, National Research Council (CNR), Padova 35121, Italy
* Correspondence: pierre.theurey@unipd.it (P.T.); paola.pizzo@unipd.it (P.P.); Tel.: +39-049-827-6067 (P.P.)

Received: 26 October 2017; Accepted: 4 January 2018; Published: 9 January 2018

Abstract: Mitochondrial dysfunction is a central event in many pathologies and contributes as

well to age-related processes. However, distinguishing between primary mitochondrial dysfunction
driving aging and a secondary mitochondrial impairment resulting from other cell alterations remains
challenging. Indeed, even though mitochondria undeniably play a crucial role in aging pathways at
the cellular and organismal level, the original hypothesis in which mitochondrial dysfunction and
production of free radicals represent the main driving force of cell degeneration has been strongly
challenged. In this review, we will first describe mitochondrial dysfunctions observed in aged tissue,
and how these features have been linked to mitochondrial reactive oxygen species (ROS)–mediated
cell damage and mitochondrial DNA (mtDNA) mutations. We will also discuss the clues that led to
consider mitochondria as the starting point in the aging process, and how recent research has showed
that the mitochondria aging axis represents instead a more complex and multifactorial signaling
pathway. New working hypothesis will be also presented in which mitochondria are considered at
the center of a complex web of cell dysfunctions that eventually leads to cell senescence and death.

Keywords: aging; mitochondria; ROS; MFRTA; mitochondrial function; mtDNA mutations; mutator
mouse; lifespan

1. Introduction
On average, a healthy person lives 80 years and one of the highest risk factors known for most
human diseases and mortality is aging. Despite the utmost importance of this observation, the origin
and mechanism of aging remains unclear. To this regard, substantial advances have been made through
investigations performed in cells and in vivo in lower organisms, such as C. elegans and D. melanogaster,
as well as in mice. The former models offer extensive experimental advantages for these studies,
i.e., shorter lifespan and easier genetic manipulation.
Many evolutionary and mechanistic theories have been elaborated on, trying to explain why and
how living organisms age. For instance, the “somatic mutation” theory suggests that aging is due
to the increase other time in DNA damage and somatic mutation, while the “telomere loss” theory
postulates a decline in cellular division capacity with age, linked to the progressive shortening of
telomeres in somatic tissues. The “altered proteins and waste accumulation” theory rather advocates
an accumulation over time of damaged proteins, protein turnover being essential to preserve cell
function and the accumulation of altered proteins contributing to a range of age related disorders [1,2].
However, from a mechanistic point of view, among all the theories, those that see mitochondria as
main actors occupy a particular place. Indeed, mitochondria have been at the center of one leading
hypothesis for 50 years: the free radical theory [3]. Even though the scientific community has shifted
to a more complex view to explain aging, embracing a network of events, mitochondria remain of high
importance because of their central position in cell homeostasis of almost every tissue. Thus, as far
as the description of molecular and cellular mechanisms are concerned, mitochondria have been
shown to participate in every main aspect of aging: decline of stem cell functions, cellular senescence,

Genes 2018, 9, 22; doi:10.3390/genes9010022 www.mdpi.com/journal/genes

Genes 2018,9,9,2222
Genes2018, 2 of 1312
1 of

shown to participate in every main aspect of aging: decline of stem cell functions, cellular senescence,
“inflammaging”
“inflammaging” and
andmany
many others [4,5]
others (Figure
[4,5] 1).1).The
(Figure Therole
roleofofmitochondria
mitochondriaininmetabolic
metabolicpathways
pathwaysisis
also
also particularly important. Their modulation, such as with caloric restriction and aerobicexercise,
particularly important. Their modulation, such as with caloric restriction and aerobic exercise,
ininturn, represents interventions than can increase lifespan [6,7] (Figure 1). Before any mechanistic
turn, represents interventions than can increase lifespan [6,7] (Figure 1). Before any mechanistic
consideration,
consideration,however,
however,thethe
involvement
involvement of mitochondria
of mitochondria in aging has has
in aging beenbeen
historically supported
historically by
supported
several reports describing decreased mitochondrial functions in aged tissue
by several reports describing decreased mitochondrial functions in aged tissue [8]. [8].

Figure1. 1.Mitochondrial
Figure MitochondrialHealth
Healthatatthe
thecenter
centerofofa aCause–Consequence
Cause–Consequencecell cellcrossroad.
crossroad.The
Theoriginal,
original,
simplistic view of the Mitochondrial Free Radical Theory of Aging (MFRTA), postulating a a
simplistic view of the Mitochondrial Free Radical Theory of Aging (MFRTA), postulating
mitochondrialactivity/ROS/mtDNA
mitochondrial activity/ROS/mtDNA isolated
isolated interaction,
interaction, was
was progressively
progressively replaced
replacedbybya amore
more
integrative view in which healthy mitochondria are the result of multiple cellular
integrative view in which healthy mitochondria are the result of multiple cellular pathways and pathways and
activities,impacting
activities, impactingdifferent
differentaspects
aspectsofofaging,
aging,inindiverse
diversetissues
tissuesand
andinin different
different manners.See
manners. Seetext
text
for details.
for details.

2.2.Mitochondrial
MitochondrialDysfunction
DysfunctionIsIsAssociated
Associatedwith
withAging
Aging
Mitochondrialalterations
Mitochondrial alterationshave
havebeen
beenextensively
extensivelydescribed
describedininagingagingtissues
tissuesofofmany
manyorgans
organsfor
fora a
long time [9]. It has been particularly studied in muscle and heart [8] and sarcopenia
long time [9]. It has been particularly studied in muscle and heart [8] and sarcopenia and heart failure and heart failure
aretwo
are twomain
maincauses
causesofofphysical
physicaldecline
decline inin the
the elderly.
elderly. In
In particular,
particular, inin these
these two
twotissues,
tissues,but
butalso
alsoin
in others like liver, brain and adipose tissue, mitochondrial alterations during aging are multiple.In
others like liver, brain and adipose tissue, mitochondrial alterations during aging are multiple.
Inparticular,
particular,the
thenumber
number andand density
density ofof mitochondria
mitochondria[10–12],
[10–12],as aswell
wellasasmitogenesis
mitogenesis[13,14],
[13,14],have
havebeen
been
showedtotobebereduced,
showed reduced,whereas
whereasfor formitochondrial
mitochondrialdynamics
dynamicsand andcontent
contentcontradictory
contradictoryinconclusive
inconclusive
results have been reported [15,16] (see also [8] for a recent review). Importantly,
results have been reported [15,16] (see also [8] for a recent review). Importantly, mitochondrial function mitochondrial
function
has has beenreported
been regularly regularly reported
to be impaired to in
bedifferent
impaired in different
aging tissues, inaging
termstissues,
of ATP in terms of and
production ATP
production and respiratory chain (RC)
respiratory chain (RC) capacity/activity [17–20]. capacity/activity [17–20].
AAkeykeyreported
reportedfeature
featureofofaging
agingmitochondria
mitochondriawas wasthe theincrease
increaseininsomatic
somaticpoint
pointmutations
mutationsand and
largedeletions
large deletionsininthe
themitochondrial
mitochondrialDNA DNA(mtDNA)
(mtDNA)[21–24].
[21–24].Interestingly,
Interestingly,these
thesemtDNA
mtDNAmutations
mutations
have been shown to be responsible for mitochondrial dysfunction [23]. Since
have been shown to be responsible for mitochondrial dysfunction [23]. Since mtDNA is located mtDNA is located very
close to the major source of reactive oxygen species (ROS) [25], oxidative damages have been
considered the main cause of mutations in mtDNA [26,27]. Indeed, the Mitochondrial Free Radical
Genes 2018, 9, 22 3 of 13

very close to the major source of reactive oxygen species (ROS) [25], oxidative damages have been
considered the main cause of mutations in mtDNA [26,27]. Indeed, the Mitochondrial Free Radical
Theory of Aging (MFRTA) considers the oxidative damage of mtDNA as the primary event affecting RC
proteins, inducing its dysfunction and increasing ROS production in a vicious cycle [3,28]. This theory
was supported by a wealth of correlative data. Indeed, oxidative stress increases with age [29–31]
and its reduction, by decreasing oxygen concentration or overexpressing in primary fibroblasts the
extracellular or intracellular form of the copper-zinc (CuZn) antioxidant superoxide dismutase (SOD),
increases cell lifespan and reduces senescence in vitro [32–34]. Furthermore, the downregulation of
the same enzyme has been shown to induce cell senescence [35]. In vivo, intracellular CuZnSOD
overexpression, together with catalase, increases the lifespan in drosophila [36]. An increase of
longevity was also reported in mice upon the overexpression of the mitochondrial manganese SOD [37]
or a catalase targeted to mitochondria [38]. Interestingly, overexpression of the extracellular form
of CuZnSOD was shown to protect mice against aging-induced cognitive impairment [39] without
increasing lifespan [40]. Missing the evaluation of mitochondrial function in these studies, the origin
of the discrepancies between the results from intracellular and extracellular SOD remains unclear and
further investigations would be required to solve the issue. Even though mitochondrial dysfunction has
been clearly established in aging tissues, the majority of the studies remained exclusively correlative
and, importantly, the observed RC decline could be secondary, for example, to hormonal alterations or
reduced physical activity of the aging organism [41–43].
However, the MFRTA remained arguably the main theory for aging for many decades. Yet,
this theory has been strongly challenged [44–46] and the scientific community had to adjust working
hypothesis to fit with a more complex mitochondria-centered network of aging mechanisms. One of the
key actors of this transition came from the creation of a fast-aging animal model, the “mutator” mouse.

3. From the Mitochondrial Free Radical Theory of Aging to a More Complex View
The mutator mouse was created in 2004 by Nils-Göran Larsson’s group, to assess the role of
mtDNA mutations and oxidative stress in aging [47]. Afterward, another group developed a very
similar model with the same D257A mutation [48]. The mouse carries a homozygous knock-in mutation
for a proof-reading deficient catalytic subunit of the mtDNA polymerase, leading to an extensive
increase in point mutations and deletions in mtDNA. Importantly, the mouse displays a reduced
lifespan and premature onset of aging-related phenotypes, providing for the first time a causative link
between mtDNA mutations and aged phenotypes [47]. Moreover, experimental evidence indicated
that the mouse phenotypes, typical of progeria (a pathology characterized by an extremely fast aging),
were driven by mtDNA alterations [48–50].
The mutator mouse, however, did not display any increased oxidative stress [48,51] even though
the animal clearly exhibited substantial mitochondrial dysfunction [50,51], shedding doubts on a
direct link between mtDNA mutations, ROS and mitochondrial alterations. Similarly, the N-terminal
methyltransferase 1 (NRMT1) knockout mouse exhibits decreased mitochondrial function and
phenotypes of premature aging, despite reduced ROS generation [52]. In addition, a study reported a
lack of gain in longevity in mice overexpressing the mitochondrial manganese SOD (MnSOD), despite
decreased oxidative stress and age-related decline in mitochondrial ATP production [53]. Moreover,
further genetic investigations in aged individuals showed that the evolution rate of the mitochondrial
mutation load was incompatible with a ROS-mediated mechanism that would predict a mutational
burst not observed experimentally [7,54]. Instead, recent works suggested that de novo mutations
come rather from DNA polymerase errors [55,56], or because of clonal expansion of original maternally
inherited mutations [7,57,58]. The role of mtDNA mutations in the mutator mouse was furthermore
challenged by different observations: (i) mtDNA mutations were order of magnitudes higher in
the model animal than in aged human tissues [59]; (ii) the mutation load of corresponding control
normal-aging mice were still higher than that in aged humans [60]; (iii) the heterozygous knock-in
mutator mouse exhibited a major increase in mutation load, compared to that of aged wild type mice,
Genes 2018, 9, 22 4 of 13

with no phenotype of premature aging [61]. Additionally, concerns have arisen regarding the type of
mutations accumulated in the mutator mouse, which did not include conical mtDNA deletion observed
in humans [62]. Another important challenge to the mtDNA mutation-mediated theory of aging came
from another mouse model. Indeed, the “deleter” mouse, which expresses a mutant-dominant version
of a mitochondrial replicative helicase, exhibits accumulation of large-scale mtDNA deletions and
mitochondrial dysfunctions, leading to mitochondrial myopathy without displaying premature aging
phenotype [63].
All these findings found echo in studies questioning whether somatic mutations in mtDNA
could ever reach a level high enough to have a significant physiological impact on mitochondrial
function [63]. Indeed, among the natural mtDNA heteroplasmy, a pathogenic mutation would need to
rise up from 60% to over 95% of level to have a functional impact on the RC [64–66]. This phenomenon,
called mitochondrial threshold, led many scientists to doubt the real implication of mtDNA mutations
in the aging process. However, an alternative hypothesis of a random mitotic segregation of mutated
mtDNA, called relaxed replication of mtDNA [67], would implicate the formation of a mosaic cell
distribution of mitochondrial deficiency in the aged tissue, which would actually fall in line with many
experimental observations [23,56,68].
Recently, it has been demonstrated that the progeria-like phenotype of the mutator mouse derives
from a somatic stem cells (SSCs) dysfunction, leading to defects in self-renewal of old post-mitotic
tissues [69]. The SSCs defect is already present at the embryonic state and precedes RC malfunctioning,
which arises only when the aging phenotype is already present in the mouse, strongly suggesting that
the SSCs dysfunction represents an early event in the mechanisms leading to aged phenotypes.

4. A Complex and Mitochondria-Centered View of Cell Dysfunctions in Aging

One of the main topics of investigation in the aging field is the link between SSCs dysfunction
and mitochondrial decline in aged tissue [4]. Indeed, stem cell exhaustion is a phenomenon that has
been described in many tissues as part of the aging process [70–72]. One of the first indications of the
role played by mitochondria in the SSCs decline was the observation that in the mutator mouse SSCs
dysfunctions could be partially reversed by antioxidants [69]. Moreover, the efficiency of somatic cells
reprograming into induced pluripotent stem cells (iPSC) was severely impaired in the animal, in a
ROS signaling-dependent manner [73]. Interestingly, the SSCs decline is relying on point mutation
accumulation in their mtDNA since the deletor mouse model, which displays mtDNA deletion only
in post-mitotic tissues, does not display SSCs renewal impairment [69]. These observations fall in
line with the idea that, in general, mitochondria are important in the metabolic control of stem cells
pluripotency through specific mitochondrial metabolites, such as α-ketoglurate [74] or NAD+ [75].
The importance of mitochondria in maintaining stem cells functions could also be suggested by the fact
that these cells have a unique capacity to eliminate old mitochondria through increased mitophagy [76]
and specific segregation of old mitochondria [77]. However, it is important to keep in mind that SSCs
decline alone does not recapitulate all the features of physiological aging [78], and that mitochondria
can affect many other related cellular phenomena, such as senescence.
Indeed, as for stem cells differentiation, senescence is characterized by profound metabolic
changes [79] and induction or repression of senescence goes through metabolic regulation. For example,
though the context might be different from aging, cell senescence mediated by oncogenes, such as BRAF
and p53, involves the modulation of mitochondrial enzymes and metabolism [80]. Other interesting
elements for understanding the complex relationship between aging and mitochondrial metabolism
come from yeasts, where it has been shown that the overexpression of the mitochondrial enzyme
malate dehydrogenase increases lifespan [81,82]. Moreover, from large scale screenings, many genes
coding for tricarboxylic acid cycle (TCA) enzymes have been identified as potential regulators of yeast
replicative lifespan [83]. These findings are supported by the ability of malate and fumarate to extend
lifespan in C. elegans [84], despite the lack of experimental evidence in mouse models. Mitochondrial
metabolism is also a key factor in nutrient sensing (NS) mechanisms [85] that are crucial regulators of
Genes 2018, 9, 22 5 of 13

lifespan. Indeed, the ability of caloric restriction (CR) to increase lifespan has been well established
and this latter effect has been shown to go through mitochondrial metabolism-mediated NS. Many
studies in yeast, and in a wide range of multicellular lower and higher organisms, have shown that CR
simultaneously increases lifespan while improving mitochondrial activity [86–88] and mitochondrial
biogenesis [89], with peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-lα)
and sirtuin 1 (SIRT1) being the two main regulators of the process identified so far [90,91]. Interestingly,
the inhibition of NS signaling pathways, such as the Insulin/IGF-1 [92] and mechanistic target of
rapamycin (mTOR) [93,94] pathways, led to similar results. Moreover, it was further showed that ROS
signaling and adenosine monophosphate (AMP)-activated protein kinase (AMPK) activation could be
the common mechanism linking CR, inhibition of NS pathways and mitochondrial activity [95,96].
The view of ROS as signaling molecules in the cellular antioxidant pathway, rather than
deleterious byproducts, led to the concept of mitochondrial hormesis (mitohormesis), corresponding
to the idea that an increase in ROS production can eventually induce an adaptive response that
will overcome the increase of oxidative stress and, eventually, has beneficiary effects [97]. This is
particularly true during aerobic exercise, another key intervention than can positively influence aging,
which increases ROS production while simultaneously improving mitochondrial biogenesis, function,
and metabolic health in the elderly [98–100]. Similarly, the link between mitochondrial function
and lifespan is complex, and cannot be oversimplified to the idea that highly active mitochondria
increase lifespan [89]. Indeed, studies have demonstrated that mild reduction of mitochondrial
function can counter-intuitively increase lifespan in yeast, worms, flies and mice. In particular, this
phenomenon was extensively studied in C. elegans, in which genetic manipulation of genes encoding
RC components, to induce mild mitochondrial dysfunction, underlies some lifespan-extending
programs [101–103]. This line of evidence has been further supported in different long-lived mouse
models [104]. This opposite association of mitochondrial activity and lifespan was shown to involve
compensatory mechanisms related to ROS signaling [105] and the mitochondrial unfolded protein
response (UPRmt ) [102,106]. The UPRmt is an adaptive stress response to depletion in mtDNA or
accumulation of unfolded proteins in the mitochondria that triggers a complete adaptive response
including a nucleus-mitochondria and inter-organs cross talk for the induction of chaperone protein
expression [107], ROS defense and metabolic adaptation. The UPRmt is the proof of the utmost
importance of mitochondrial homeostasis for cell health since more than 400 genes are involved
to activate numerous maintenance pathways in the case of mitochondrial distress, to stabilize and
recover their function. A subtle equilibrium constitutes a vast mitochondrial surveillance system to
monitor the health of the mitochondrial pool [108]. However, the relationship between mitochondrial
dysfunction, UPRmt and longevity is complex, as it appears that UPRmt activation, alongside multiple
pathways activated during mitochondrial dysfunction, is required but not sufficient for gaining
longevity [108,109]. Another major mechanism in maintaining mitochondrial protein homeostasis
is mitoproteases, proteolytic mitochondrial enzymes important in protein quality control and in
preventing accumulation of misfolded proteins. Interestingly, mitoproteases overexpression increases
lifespan, even though these experiments were performed only in a fungal model of aging [110,111].
When the UPRmt is overwhelmed, coordination of mitogenesis and mitophagy, which is the
selective removal of damaged mitochondria by autophagy, ensures a fresh and functional pool of
mitochondria sustaining cellular function [112]. Even though Parkin (a key protein in mitophagy; [113])
overexpression or suppression, respectively, increase or decrease lifespan in drosophila [114,115], direct
evidence that mitophagy can modulate aging in other organisms is missing [4]. However, some clues
indicate that mitophagy can modulate the effect of lifespan-controlling pathways. For example, it has
been reported that the knockdown of mitophagy-related proteins decreases lifespan of mutant or
CR long-lived C. elegans [116,117]. Moreover, Parkin overexpression attenuates aging-related muscle
atrophy in mice [118], while its ablation decreases lifespan [119].
Thus, it appears that maintaining healthy mitochondria, not necessary active, is the central
elements for healthy aging and long lifespan. The utmost importance of preserving the integrity of
Genes 2018, 9, 22 6 of 13

the mitochondrial pool is also sustained by the fact that mitochondria-derived damage-associated
molecular patterns (DAMPs), in particular free mtDNA molecules and formylated peptides, liberated
upon physical damage to mitochondria, can induce inflammatory response through multiple
pathways [120–122]. Importantly, chronic inflammatory state is one of the hallmark of aging and
has been termed “inflammaging” [123]. This process directly participates to the physical decline
in the elderly, as illustrated by the fact that, in mice, ablation of the Nlrp3 inflammasome protects
against age-related pathologies [124]. Moreover, mitochondria have been shown to mechanistically
participate to the antiviral immune response: indeed, the innate immunity adaptor protein MAVS has
been shown to be localized at the mitochondrial outer membrane [125]. Altogether, these findings
strongly support the idea of an intimate relationship between mitochondria and the inflammatory
process, in which the activation of this latter induces mitochondrial impairment and vice versa.
Accordingly, many inflammatory pathologies have been associated to mitochondrial defects [126,127].
The importance of mitochondrial health in inflammation, one of the main biological response in
maintaining organism homeostasis, is another example of the key role played by this organelle in the
eukaryotic cell in general, beyond aging pathways.

5. Conclusions
Human lifespan has increased drastically in the past decades and is likely to keep raising [127,128].
Increasing number of people at old and very old ages will pose major challenges for healthcare systems
and, in this context, the promotion of healthy aging is crucial. To this regard, the better knowledge of the
molecular mechanisms behind the aging process is a main priority of modern societies. Unfortunately,
they are still unclear and the current working models are of increasing complexity [129]. In the past
and in the current hypotheses, mitochondria had, and still have, a key position due to their central role
in eukaryotic cells/tissues, in almost every form of homeostasis (Figure 1). Even though mitochondrial
impairment is clearly associated with aging, the high complexity of aging phenotypes, and their
underlying molecular mechanisms, make the deciphering of the real causing elements difficult [5].
Moreover, the discovery of mitohormesis in stress response and ROS signaling pathways nuanced
the idea of active and healthy mitochondria, and of ROS production and oxidative stress. Indeed,
as described above, increased ROS and less active mitochondria can promote healthy aging and
long lifespan. Even in this complex scenario, more than ever, acting on mitochondria seems to be
an attractive perspective in order to achieve gain in health and lifespan, since rejuvenating aged
mitochondria could be an interesting therapeutic strategy to improve health in the elderly (Figure 1).
To this regard, the best interventions identified so far in mammals and different aging models remain
dietary intervention [130–132] (i.e., caloric restriction, intermittent feeding, nutrient deprivation or
other fasting-mimicking diets) and physical activity [133].

Acknowledgments: The authors thank grants from the University of Padova; the Italian Ministry of University
and Scientific Research and EU Joint Programme in Neurodegenerative Disease, 2015–2018, “Cellular Bioenergetics
in Neurodegenerative Diseases: A system-based pathway and target analysis” for their research work support.
P.T. is a Research fellow of the EU Joint Programme in Neurodegenerative Disease grant.
Conflicts of Interest: The authors declare no conflict of interest.

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